Australia has never permitted fluoroquinolone use in food-producing animals. We examined local retail poultry for contamination with fluoroquinolone non-susceptible Escherichia coli, then explored the hypothesis that their presence may be due to co-selection of resistance determinants. Between August and November 2010, samples from 30 locally produced, uncooked retail poultry carcasses from four different processing centres underwent selective enrichment culture for ciprofloxacin non-susceptible E. coli. Their chromosomal- and plasmid-mediated resistance determinants were characterized, and phylogenetic analysis and transformation experiments were performed. Unexpectedly, we found nine (30 %) of our small collection of poultry samples carried fluoroquinolone non-susceptible E. coli of which nearly half possessed aac(6′)-Ib-cr, a novel plasmid-mediated gene encoding an aminoglycoside acetylating enzyme that also confers fluoroquinolone resistance. All nine isolates were co-resistant to amoxicillin, gentamicin, tetracycline and trimethoprim/sulfamethoxazole - all antibiotic classes that are registered for use in poultry reared for food production within Australia. Their unique phylogenetic relatedness suggested clonal dissemination driven by non-fluoroquinolone selective pressures. aac(6′)-Ib-cr was successfully transformed and selected for using non-fluoroquinolone antibiotic pressure. Vertical and perhaps horizontal co-selection may be contributing to the emergence of fluoroquinolone resistance in poultry and could play a similar role in the human setting. This suggests that preservation of the usefulness of fluoroquinolones may require more than just restriction of their use in isolation from other interventions. © 2013 SGM.